The lethal factor (LF) produced by toxigenic strains of Bacillus anthracis is a Zn²⁺-endopeptidase that cleaves the mitogen-activated protein kinase kinases (MAPKKs) MEK1, MEK2 and MKK3. Using genetic and biochemical approaches, we have extended the study of LF proteolytic specificity to all known MAPKK family members and found that LF also cleaves MKK4, MKK6 and MKK7, but not MEK5. The peptide bonds hydrolysed by LF within all MAPKKs were identified. Cleavage invariably occurs within the N-terminal proline-rich region preceding the kinase domain, thus disrupting a sequence involved in directing specific proteinŐprotein interactions necessary for the assembly of signalling complexes. Alignment of the sequences flanking the site of cleavage reveals the occurrence of some consensus motifs: position P2 and P1´ are occupied by hydrophobic residues and at least one basic residue is present between P4 and P7. The implications of these findings for the biochemical activity and functional specificity of LF are discussed.